370 BIOCHIMICA ET BIOPHYSICA ACTA VOL, 26 (I957)

T H E N U C L E A R MAGNETIC R E L A X A T I O N T I M E OF W A T E R

P R O T O N S IN F E R R I H E M O G L O B I N S O LU TIO N S

NORMAN DAVIDSON* AND R O N A L D GOLD

Department o] Chemistry, Harvard University, Cambridge, Mass. (U.S.A.)

The purpose of this work has been to at tempt to determine the position of the iron atoms of ferrihemoglobin with respect to the surface of the protein by measuring the relaxation time of water protons in ferrihemoglobin solutions.

Paramagnetic ions are effective in producing spin-lattice relaxation of the proton magnets in an aqueous solution 1-3. The magnetic field exerted by the paramagnetic ion on the proton fluctuates due to the random thermal motions of the two species. This results in a decrease in the proton magnetic relaxation time (T1) and in a line- broadening effect (/Iv = I/aT1). Under suitable conditions, this relaxation effect is proportional to /£2p/Zi2on G/a O (/~p and/*ion are the magnetic moments of proton and ion, c is the concentration of the paramagnetic ion, D is the diffusion coefficient of the most rapidly diffusing species, and a is the closest distance of approach of a water proton and the paramagnetic ion 2. It is therefore expected that increasing a by sur- rounding the ion with a complexing ligand will decrease the spin-lattice relaxation effect of the ion. This effect has been observed for various complexing agents and paramagnetic ions in previous work 4, 5. In the course of the present experiments, it was incidentally observed that phosphate, pyrophosphate and EDTA decrease the line-broadening effects of Fe +3 ions on water protons. I t must be emphasized that at present it is not possible to make a quantitative determination of a from such measurements because of various inadequacies in the theory. Probably the main uncertainty is related to the spin-lattice relaxation time of the paramagnetic ion i tself-- i f this time is shorter than or comparable to the typical time for relative diffusion of the ion and a water molecule past each other (~a~/6D) the quantitative relation stated above is not applicable.

METHODS

Measurements were made wi th a Varian High Resolut ion NMR Spectrometer at a frequency of 4 ° mc and field s t rength of 940o gauss. Al though the in s t rumen t is not designed for the purpose, fairly sat isfactory measurements can be made for re laxat ion t imes greater than 2 sec by sa tura t ing the spin sys tem with a large radiofrequency signal ( ~ o . o o i gauss), and observing the growth of the NMR absorpt ion signal on the recorder when the RF dial is quickly switched to its lowest set t ing ( t~o .oooo 5 gauss). Modulation frequencies and ampli tudes of ca. 2 or i sec -1 and o.030 gauss were used; the water proton peak was less t han o.ooi gauss wide.

The relaxation t ime of oxygen free water is about 3.2 sec. In order to decrease this p r o t o n - p ro ton interact ion so tha t effects due to paramagnet ic solutes could be observed, solutions in mix tures of H20 and D~O were used e. The solutions were all degassed by bubbl ing nitrogen.

* Pe rmanen t address: Dept. of Chemistry, California Ins t i tu te of Technology, Pasadena, Calif. (U.S.A.).

Re/erences p. 372[373 .

VOL. 26 (1957) NUCLEAR MAGNETIC RELAXATION TIME OF WATER PROTONS 371

A paste of crystals of human carbonmonoxyhemoglobin (HbCO) in 2. 5 M phosphate buffer prepared by the Drabkin procedure was kindly supplied by Dr. J. VINOGRAD. Concentrations were determined by spectrophotometry and are reported per heme group. Samples were oxidized to ferrihemoglobin (Hb +) by addition of a slight excess of K3Fe(CN)8 in D20 and the CO removed by bubbling nitrogen.

RESULTS

The principal results are as follows. For solutions of: Fe (H20)e+++, 1o-6-1o-SM; HC10,, O.OlM; in I : 9 H ,O:D20 ; 1 / T a = 1.2 (-4- 0.2) • lO4C + 0.07 sec -a (c ---- concen- tration of Fe +++ in mole liter-a).

By line-broadening experiments, it is observed that the EDTA chelate of Fem is about I / 7 as effective as Fe +++ in relaxing water protons.

For solutions of ferriheme chloride (IO-4-4 • Io-4M) in pyridine, I / T 1 ~- 8 (::k 2) .

io * c + 0.07 sec -a. (Corresponding line-broadening effects of ferriheme on the fine structure of the pyridine NMR spectrum were observed.)

A typical hemoglobin experiment is as follows 1. 9 g HbCO paste plus I ml H20 were dissolved in D,O to give 25 ml of solution in which Cnb~o = I. 5.1O-3M. The sample was oxidized to ferrihemoglobin (Hb+); excess NaCN ( ,~o.oIM) was added to make ferrihemoglobin cyanide (HbCN). Relaxation times for these solutions and for I :1 dilutions with D,O were measured,

c HbCO Hb + HbCN

7.o. i o - t M lO. 7 6. 3 11. 7 1.4" I ° -SM 8-7 4 9.4 sec

The HbCO and HbCN solutions have relaxation times somewhat greater than ex- pected for their light water content; either an impurity or the protein itself con- tributes somewhat to z / T v HbCN has a static magnetic moment corresponding to one unpaired electron ; its predicted effect would be quite small. I t should be recalled tha t Fe (CN)6 -3 with a static moment due to one unpaired electron has an effective/~ for proton relaxation of < o.I, because of electron paramagnetic relaxation phe- nomena.

I t is therefore clear that ferrihemoglobin has an additional relaxing effect on water protons that is expressed by the equation I / T 1 = 11o (-4- 30) c + const.

DISCUSSION

Thus, the relative efiiciencies of Fe (H~O)e +++, Fem (EDTA) -1, ferriheme, and ferri- hemoglobin, (all with static moments corresponding to 5 unpaired electrons) are as lO 4, 1.5" lO 8, 8. lO s, and 1.2. io s for water (or pyridine) protons.

While the results reported here are not very accurate, there is no doubt whatso- ever about the salient conclusion that Fe uI in methemoglobin is less effective than Fe (H,O)e+++ by a factor of about IOO and less effective than the iron in ferriheme by a factor of 5 or more.

I t is difficult to assess the extent to which these results are due to geometrical separation effects and the extent to which they are due to other factors.

The electronic state of iron in ferrihemoglobin is not quite the same as the eS s tate of Fe (HtO)e+++7; this is revealed by the markedly anisotropic g factors observed

Relerences p. 372]373.

37 2 N. DAVIDSON, R. GOLD VOL. 26 (i957~

b y p a r a m a g n e t i c resonanceS, 9. (Never the le s s , t h e p a r a m a g n e t i c r e l a x a t i o n t im~ of ferrihemog]obin crystals is reasonably long even at room temperatureS.)

The ferriheme in pyridine experiments were performed to study the effect of iron in a similar electronic environment to that of ferrihemoglobin. There arc un resolved questions as to the state of aggregation of hemin in pyridine; it is probable that it is monomeric and that the iron forms six approximately octahedral bonds to two pyridines and to the four positions of the heme ring. The relaxation effect is therefore due to the interaction of this molecular unit with additional pyridine molecules which diffuse past it. On this view, if the heme in hemoglobin were on the surface of the protein, and separated from the mobile water molecules which giw; the NMR signal by only a monomolecular layer of water of hydration, ferrihemoglobin would be expected to be I/2 as effective as hemin or more, since only one side of the molecule would see the solvent, but there would be no shielding by a bulky pyridine group. Since ferrihemoglobin is less than 1/2 as effective as hemin, it is probable that the heme group is not on the surface of the protein. A quantitative determination is not possible, but it is reasonable to assert that the paramagnetic iron atom is 5-1o A below the surface, on the basis of the data reported here. This conclusion is in accord with the theory of PAULING I° and contrary to that of KEILIN 11. It is in no way contradictory to the several studies which fix the orientation of the heme plane with respect to the crystallographic axes of the molecule 9,12.

It may be that each heme group is on the surface of a water-containing crevice of the protein. It is probable that the exchange of crevice water and external water would be sufficiently slow so that the crevice water would not contribute to the NMR signal and hence to the relaxation time.

It may be recalled that there is evidence that the iron in cytochrome c is in a crevice and that the iron in myoglobin is on the surface of the protein '3-15. Because of limited time and facilities, these substances were not investigated by us. It would also be of interest to study the effect of the dissociation of hemoglobin by acid or urea on its relaxation behavior. We believe that these and related problems provide a fruitful field of inquiry for a laboratory where equipment for the accurate measure- ment of spin-lattice relaxation times is available.

W e wish to e x p r e s s our t h a n k s to Dr. A. BOTHNER-BY for his g e n e r o u s a n d e x t e n s i v e

h e l p a n d adv ice in c o n n e c t i o n w i t h t h e o p e r a t i o n of t h e N M R m a c h i n e .

SUMMARY

Ferrihemoglobin molecules in solution decrease the nuclear magnetic relaxation time of water protons. Comparison of this effect with the larger effects due to Fe(H20)s +++, Fe (versene) complex, ferriheme chloride (on pyridine protons), and the smaller effects due to carbonmonoxyferro- hemoglobin and ferrihemoglobin cyanide suggests that the iron atoms of hemoglobin are situated 5-1o A below the surface of the protein.

REFERENCES

1 F. BLOCB, W. HANSEN AND M. PACKARD, Phys. Rev., 7 ° (1946) 474. z N. BLOEMBERGEN, E. PURCELL AND R. POUND, Phys. Rev., 73 (1948) 679. * A. W. NOLLE AND L. O. MORGAN, J. Chem. Phys., 26 (1957) 642. 4 B. M. KOZYREV AND A. I. RIVKIND, J. Exptl. Theoret. Phys., 27 (1954) 69.

VOL. 26 (1957) NUCLEAR MAGNETIC RELAXATION TIME OF WATER PROTONS 373

L. O. MORGAN, A. W. I~IOLLE, R. L. HULL AND J. MURPHY, J. Chem. Phys., 25 (1956) 206. 6 W. A. ANDERSON AND J. T. ARNOLD, Phys. Rev., ioI (1956) 511.

J. S. GRIFX=ITH, Proc. Roy. Soc. (London), A235 (1956) 23-36. 8 D. J. E. I N G ~ AND J. E. B R SN Srr , Discussions Faraday Sot., 19 (1956) 14o. g J. E. BlZNNETT, J. F. GIBSON AND D. J. E. INGRAM, Proc. Roy. Soc. (London), A24o (1957) 67.

1.0 R. C. C. ST. GEORGF* AND L. PAULING, Science, 114 (1951) 2972. Xl D. KEILIN, Nature, i7I (1953) 922. xs D. J. E. INGRAM, J. F. GmSON AND M. F, PERU'rZ, Nature, 178 (i956) 9o6. as H. THEORELL, J. Am. Chem. Soc., 63 (1941) 182o. 14 p. GEORG~ AND G. I. H. HANANIA, Nature, 175 (1955) lO34. xs j . C. KENDREW AND R. G. PARRmH, Nature, 175 (1955) 2o6.

Received June 27th, 1957

DIE BINDUNG DES ERSCHLAFFUNGSFAKTORS

VON MARSH AN DIE MUSKELGRANA

HILDEGARD PORTZEHL

Institut ]~r Physiologie im Max-Planck-Institut tar Medizinische Forschung, Heidelberg (Deutschland)

I

Seit der Entdeckung eines physiologischen Erschlaffuugsfaktors in Muskelextrakten durch MARSH 1 sind zahlreiche Arbeiten erschienen, die diesen Erschlattungsfaktor zu identifizieren versuchen. In der Mehrzahl dieser Arbeiten wird die Erschlattung, die der Faktor bewirkt als eine Erschlaffung dutch Restitution des ATP (Adenosintriphosphat) angesehen. In der Arbeit yon BENDALL I wird zwar ebenfalls angenommen, dass ein ATP-restituierendes Enzym, die Myokinase, mit dem yon MARSH entdeckten Faktor identisch sei, aber es wird nicht angenommen, dass die Erschlaffung auf der Myokinase-Wirkung beruhe, sondern durch einen anderen Mechauismus des Myokinase- Proteins bewirkt w~irde. Sehr intensive japanische StudienS, 4 fiber die Natur des Erschlaffungs- faktors kommen zu dem Ergebnis, dass der Faktor aus meheren Komponenten zusammengesetzt sei.

Es ist seit langem bekannt, dass der ungereinigte Erschlaffungsfaktor im Muskelextrakt unter allen Bedingungen unter denen er die Kontraktion riickg'~ngig macht oder verhindert auch die ATP-Spaltung dutch das Aktomyosin aufhebt. Es ist bisher noch hie bewiesen und teilweise sogar widerlegt, dass die ATP restituierenden Substanzen, die yon den verschiedenen Autoren als Erschlaffungsfaktor angesprochen werden, ebenfalls die ATP-Spaltung auiheben. Die einzige entgegenstehende Angabe yon BENDALL 2 fiber die hemmende Wirkung der Myokinase auf die ATP-Spaltung kann yon I~RRY 6 nicht best~tigt werden. Es muss iniolgedessen die Hemmung der ATP-Spaltung dutch den Erschlaf/ungsfaktor als eine eindeutigere Eigenschaft des Faktors angesehen werden als die Erschlaitung yon Muskelmodellen, Daher wird die Spaltungshemmung in dieser Arbeit zur Charakterisierung des Erschlaffungstaktors benutzt.

II

Alle Autoren die sich bisher mit der Natur des Erschlaffungsfaktors besch~ftigt haben, nehmen an, dass dieser Faktor ganzl,*,s, ~ oder teilweise 4 zum sarkoplasmatischen System des Muskels geh6rt. Es wird im folgenden gezeigt, dass griindlich gewaschene isolierte Muskelfibrillen allein dutch die Zugabe von Muskelgrana an der ATP- Spaltung verhindert werden.

Wird der in iiblicher Weise hergestellte Rohextrakt des Muskels dadurch auf seine Wirkung als Erschlaffungsfaktor quantitativ gepriift, dass er den Fibrillen in Literatur S. 377.